Evidence already gathered in our laboratory (see Research Plan) clearly indicates the efficacy of examining the effects of the four major classes of drugs of abuse on the mechanisms of synaptic transmission. Our work will be concerned with the modes by which these drugs alter the phenomena of neurotransmitter release and binding to post synaptic membrane sites. A systematic study of possible interactions of psychoactive agents with the synaptic substructures involved in presynaptic storage and release of four major neurotransmitters and with the respective high affinity binding sites on synaptosomal membranes promises to yield considerable knowledge with respect to the role of various components involved in chemical transmission as well as the mechanism of action of the drugs of abuse. We propose to isolate synaptic vesicles, synaptosomal membranes, and junctional complexes using differential centrifugal and density gradient techniques. The homogeneity of these materials will be established by determining the specific activities of known marker enzymes and electronmicroscopy. The effects of psychoactive drugs on neurotransmitter release will be determined indirectly by the assay of the Mg ions dependent ATPase activity associated with synaptic vesicles and directly employing a flow dialysis system. We will further characterize the high affinity binding of neurotransmitter substances to synaptosomal membranes by equilibrium dialysis and examine the influence of drugs of abuse in this system. In addition, we will monitor the interactions between synaptosomal membranes and both transmitter substances and psychoactive drugs by measuring membrane conformational changes using fluorescent probe techniques.